Detection of microorganisms is routinely performed in testing environmental and biological samples. For example, as a public health protection measure, many countries mandate that utility providers monitor recreational and potable water for microorganisms that are indicators of faecal contamination. Faecal contamination of water bodies in urban environments arises from inadequate waste management in, for example, developing countries, or from failure of water management systems, such as leakage from compromised or overburdened sewerage networks. Faecal contamination can also occur in drinking water distribution systems from sewage or drainage ingress when water mains experience transient depressurization, such as during maintenance or pump failure. There is a link between exposure to human waste and the transmission of infectious diseases such as cholera, typhoid fever, shigellosis and acute gastroenteritis. Considerable effort is expended in detecting, quantifying and tracking instances of faecal contamination of water bodies to ensure drinking water meets sanitary guidelines and that recreational waters remain safe for primary and secondary contact activities such as swimming and boating respectively. The monitoring of microbial water quality is routinely undertaken by or on behalf of, local authorities or utilities who will use the information to control public access to recreational facilities and water resources.
Usually, faecal indictor bacteria (FIB) are monitored as a proxy for faecal contamination of water, traditionally relying on the enumeration of faecal coliforms (FC), usually understood to mean E. coli, and total coliforms (TC), to assess microbial water quality. FIB, not necessarily disease causing themselves, must fulfil a number of criteria, including low tendency to proliferate in the hydrosphere, to be nonpathogenic, to persist longer than pathogens in the hydrosphere, and to have known provenance of either human or animal origin (Nshimyimana et al., J. Appl. Microbiol. 2014; 116(5):1369-83; Cabral, Int. J. Environ. Res. Public Health. 2010; 7(10):3657-703). Alternative indicators, such as Enterococcus faecalis, Clostridium perfringens and Bacillus spp. have also been used as FIB in an attempt to more definitively identify human faecal contamination of water (Douterelo et al., Water Res. 2014; 65:134-156; U.S. patent application Ser. No. 11/428,046). There are a number of drawbacks to using FIB as a proxy for detection of faecal contamination many of which stem from the indirect nature of the procedure, the length of time required for growth, and the assumptions underlying FIB selection. Recently, molecular advances have allowed the monitoring of emerging faecal indicators which were previously impossible with culture based techniques, such as real time PCR detection of the uidA gene or the human specific bacteroides HF183 marker (Nshimyimana et al., J. Appl. Microbiol. 2014; 116(5):1369-83).
While it is true that specificity and detection time can be improved by molecular techniques, their direct correlation to pollution quantity remains unsatisfactorily resolved and hence does their application to improve risk based decision making. For example, PCR consistently underestimates the proportion of bifidobacterium when compared with culture based methods, which results in positive detection of organisms that are not detected by regulatory approved culture based techniques (Cabral, Int. J. Environ. Res. Public Health. 2010; 7(10):3657-703).
Modern enzymatic assays and many molecular and traditional culture techniques for the enumeration of E. coli or FC exploit the uidA gene which encodes for the β-glucuronidase enzyme and which almost 98% of E. coli possess (Martins et al., Appl. Environ. Microbiol. 1993; 59(7):2271-6; Manafi et al., Microbiol. Rev. 1991; 55(3):335-48). Similarly, the lacZ gene which encodes for β-galactosidase is indicative of TC, such as Klebsiella spp., Citrobacter spp., as well as E. coli (Cabral, Int. J. Environ. Res. Public Health. 2010; 7(10):3657-703; Manafi et al., Microbiol. Rev. 1991; 55(3):335-48; Bej et al., Appl. Environ. Microbiol. 1991; 57(4):1013-7). The current technology for commercial E. coli detection usually involves a uidA or lacZ specific monosaccharide conjugated to a chromogenic or fluorogenic compound (aglycon). When cleaved by the action of the corresponding enzyme found in the uidA or lacZ gene region, the optically active aglycon is released into the medium and is subsequently detected either spectroscopically, by visual assessment, by fluorimetery or inspection under a UV lamp (Manafi et al., Microbiol. Rev. 1991; 55(3):335-48; Nelis and Van Poucke, Environmental Challenges. Springer) and thus the organism of interest is presumed present.
Enzymatic, and culture based approaches may be biased, labour intensive, expensive and prone to interference from the natural properties of environmental samples, such as humic acid content, turbidity and non-target organisms (Hata et al., Appl. Environ. Microbiol. 2014; Kapoor et al., Appl. Environ. Microbiol. 2015; 81:91-99). However, the current regulatory favoured technique for evaluation of microbial water quality still remains the culture based or enzymatic enumeration of TC or FC. These approaches are predominantly laboratory-based, labour intensive and suffer from poor detection times. These shortcomings have not been satisfactorily resolved, which leaves significant opportunity for innovation to achieve faster detection at low cost (Rompréet al., J. Microbiol. Methods. 2002; 49:31-54).
Regardless of the approach, fast and automated detection of FIB that do not require specialist operators is desirable because of the cost savings that could be achieved through reduced labour costs and higher throughput. Even as the range of indicator proxies expands and the potential transduction framework and detection technology increases (Lazcka et al., Biosensors and Bioelectronics 2007; 22:1205-1217), there still remains regulatory resistance to emerging FIB detection techniques. Consequently, no regulatory guidelines exist in the EU or the US that prescribe standard protocols for the application of molecular approaches to FIB monitoring and all approved approaches are for laboratory based chromogenic, fluorogenic or traditional culture based methods (Douterelo et al., Water Res. 2014; 65:134-156).
Electrochemical detection of E. coli using glycoconjugates has been reported previously with reasonable detection times (Perez et al., Anal. Chim. Acta. 2001; 427:149-154.). Perez et al. reported the detection of E. coli at concentrations of 1 CFU mL−1 (laboratory sample) and 4.5 CFU mL−1 (marine sample) in 10 h and in 7.3 h respectively. However, in addition to a filtration step, the method described by Perez et al. utilized a combination of flow injection analysis (FIA), and a potentiostatic technique to achieve sensitive electrochemical detection of a 4-aminophenyl (4-AP) aglycon that had been cleaved from 4-aminophenyl-β-d-galactopyranoside (4-APgal) glycoconjugate by E. coli. The complexity and low throughput of this method make it incompatible with automated or remote applications as it requires skilled operation.
Low cost bioelectroanalytical methods would be amenable to automation and portable applications. However, for self-powering systems to work, the aglycon component of the detection compounds needs to be reversibly oxidized by microorganisms to achieve microbially mediated electron transfer to the electrode as E. coli is not strongly electrogenic in character (Wang et al., Phys. Chem. Chem. Phys. 2013; 15:5867-5872; Choi et al., BULLETIN-KOREAN CHEMICAL SOCIETY 2003; 24:437-440; Roller et al., J. Chem. Technol. Biotechnol. 1984; 34:3-12). Theoretically, electrochemical detection compounds could achieve more sensitive and faster E. coli detection than chromogenic equivalents because upon being cleaved, the aglycon component of the glycoconjugate detection compound is able to contribute to signal intensity many times as it may mediate numerous redox interactions between the microorganism and the electrode. Conversely, the contribution to the overall signal of a chromogenic aglycon is merely addictive. Compounds that are commonly used in coliform detection framework, such as 4-APgal and 4-nitrophenol-β-d-glucuronidase (4-NPglu) have been designed for colorimetric and fluorimetric assays. Although they have some electrochemical activity, their redox chemistry is not fully reversible by E. coli under physiological conditions or the redox reactions suffer from slow kinetics. Hence their utility in bioelectroanalytical detection is restricted to techniques described by Perez et al. that do not rely on or exploit the microbially mediated electron transfer that is achievable in bioelectranalytical systems.
Therefore, there is still need in the art for alternative methods that overcome the drawbacks of existing techniques.